The blood-brain barrier (BBB) is the major obstacle for the development of centrally active peptides. The capillaries in the brain parenchyma possess high-resistance, tight junctions between the endothelial cells. The cells also lack pores, thus the brain capillary endothelium behaves like a continuous lipid bilayer. Diffusion through this layer, the physical BBB, is largely dependent on the lipid solubility of the solute. Water-soluble molecules (for example, glucose, essential amino acids, glutamate) enter the brain almost exclusively by carrier-mediated transport. Most peptides, such as the naturally occurring enkephalins, are hydrophilic and do not cross the BBB, due to the absence of specific transport systems in the membrane. Their metabolic instability also implies that the highly active neuropeptide degrading enzymes, such as the capillary-bound aminopeptidase, arylamidase and enkephalinase, constitute an enzymatic BBB for peptides that results in their rapid cleavage. See, for example, W. M. Pardridge, in Peptide Drug Delivery to the Brain (Raven Press, New York, 1991), pp. 244-250; W. M. Pardridge and L. J. Mietus, Endocrinology 109, 1138 (1981); L. B. Hersch, N. Aboukhair, S. Watson, Peptides 8, 523 (1987); J. M. Hambrook, B. A. Morgan, M. J. Ranee, C. F. C. Smith, Nature 262,782 (1976); and J. F. McKelvy, in Brain Peptides, D. T. Krieger, M. J. Brownstein, J. B. Martin, Eds. (Wiley-Interscience, New York, 1983), pp. 117-133.
Various strategies have been applied to direct centrally active peptides into the brain. An invasive procedure that includes surgical implantation of an intraventricular catheter followed by pharmaceutical infusion into the ventricular compartment delivers a metabolically unstable peptide only to the surface of the brain; D. G. Poplack, A. W. Blayer, M. E. Horowitz, in Neurobiology of Cerebrospinal Fluid, J. H. Wood, Ed. (Plenum Press, New York, 1981), pp. 561-578. Transient opening of the tight junctions by the intracarotid infusion of an osmotically active substance (mannitol, arabinose) in high concentrations (&gt;1M) may facilitate an indiscriminate delivery of molecules that otherwise cannot cross the BBB; see E. A. Neuwelt and S. I. Rappaport. Fed. Proc. 43, 214 (1984). However, this procedure is accompanied by severe toxic effects which can lead to inflammation, encephalitis and seizures. These invasive procedures are only justified for some life-threatening conditions, and are not acceptable for less serious illnesses.
A noninvasive method for peptide delivery into the central nervous system (CNS) has been suggested that utilizes the formation of chimeric peptides [W. M. Pardridge, Endocrinol. Rev. 7, 314 (1986)]. This strategy relies on the presence of specific receptor-mediated transcytosis systems in the BBB for certain larger peptides such as insulin, insulin-like growth factor, transferrin and albumin. Covalently coupling (for example, via disulfide bonds) a non-transportable peptide to these transport vectors results in a chimeric peptide that can also undergo receptor-mediated transcytosis, and the active peptide can be released by its enzymatic cleavage in the CNS. However, these carriers are not brain-specific, as uptake by non-neural cells or cells outside the CNS has also been shown. See F. Ito, S. Ito, N. Shimizu, Mol. Cell. Endocrinol. 36, 165 (1984). Low amounts of the peptide relative to the carrier molecule, and the receptor-based cellular transport mechanism that has physiologically limited transporter capacity (saturable) also prevent pharmacologically significant amounts from entering the brain. Finally, release of the active peptide from the conjugate has not been documented.
Another method for peptide delivery is a simple pharmacologically based approach in which peptide "prodrugs" are applied that are lipophilic esters or amides of the molecule [T. Tsuzuki et al, Biochem. Pharmacol. 41, R5 (1991)]. Although the acquired lipophilicity of these prodrugs may assure penetration to the BBB (and to other membranes), this is not the sole factor involved in the transport of a peptide into the CNS. BBB transport of cyclosporin, which is one of the most lipid soluble peptides, is paradoxically low due to peptide degradation [D. J. Begley et al, J. Neurochem. 55, 1222 (1990)].
A dihydropyridine.revreaction.pyridinium redox system has recently been successfully applied to delivery to the brain of a number of drugs. Generally speaking, according to this system, a dihydropyridine derivative of a biologically active compound is synthesized, which derivative can enter the CNS through the blood-brain barrier following its systemic administration. Subsequent oxidation of the dihydropyridine species to the corresponding pyridinium salt leads to delivery of the drug to the brain.
Four main approaches have been used thus far for delivering drugs to the brain using a redox system. The first approach involves derivation of selected drugs which contain a pyridinium nucleus as an integral structural component. This approach was first applied to delivering to the brain N-methylpyridinium-2-carbaldoxime chloride (2-PAM), the active nucleus of which constitutes a quaternary pyridinium salt, by way of the dihydropyridine latentiated prodrug form thereof. Thus, a hydrophilic compound (2-PAM) was made lipoidal (i.e. lipophilic) by making its dihydropyridine form (Pro-2-PAM) to enable its penetration through lipoidal barriers. This simple prodrug approach allowed the compound to get into the brain as well as other organs, but this manipulation did not and could not result in any brain specificity. On the contrary, such approach was delimited to relatively small molecule quaternary pyridinium ring-containing drug species and did not provide the overall ideal result of brain-specific, sustained release of the desired drug, with concomitant rapid elimination from the general circulation, enhanced drug efficacy and decreased toxicity. No "trapping" in the brain of the 2-PAM formed in situ resulted, and obviously no brain-specific, sustained delivery occurred as any consequence thereof: the 2-PAM was eliminated as fast from the brain as it was from the general circulation and other organs. Compare U.S. Pat. Nos. 3,929,813 and 3,962,447; Bodor et al, J. Pharm. Sci., 67, No. 5, 685 (1978). See also Bodor, "Novel Approaches for the Design of Membrane Transport Properties of Drugs", in Design of Biopharmaceutical Properties Through Prodrugs and Analogs, Roche, E. B. (ed.), APhA Academy of Pharmaceutical Sciences, Washington, D.C., 98-135 (1976). Subsequent extension of this first approach to delivering a much larger quaternary salt, berberine, to the brain via its dihydropyridine prodrug form was, however, found to provide site-specific sustained delivery to the brain of that anticancer agent. See Bodor et al, Science, Vol. 214, Dec. 18, 1981, pp. 1370-1372. This approach is not applicable to the delivery of peptides, however, since they do not comprise active quaternary pyridinium salts.
The second approach for delivering drugs to the brain using a redox system involves the use of a dihydropyridine/pyridinium carrier chemically linked to a biologically active compound. Bodor et al., Science, Vol. 214, Dec. 18, 1981, pp. 1370-1372, outlines a scheme for this specific and sustained delivery of drug species to the brain, as depicted in the following Scheme A: ##STR1## According to the scheme in Science, a drug [D] is coupled to a quaternary carrier [QC].sup.+ and the [D-QC].sup.+ which results is then reduced chemically to the lipoidal dihydro form [D-DHC]. After administration of [D-DHC] in vivo, it is rapidly distributed throughout the body, including the brain. The dihydro form [D-DHC] is then in situ oxidized (rate constant, k.sub.1) (by the NAD.revreaction.NADH system) to the ideally inactive original [D-QC].sup.+ quaternary salt which, because of its ionic, hydrophilic character, should be rapidly eliminated from the general circulation of the body, while the blood-brain barrier should prevent its elimination from the brain (K.sub.3 &gt;&gt;k.sub.2 ; k.sub.3 &gt;&gt;k.sub.7). Enzymatic cleavage of the [D-QC].sup.+ that is "locked" in the brain effects a sustained delivery of the drug species [D], followed by its normal elimination (k.sub.5), metabolism. A properly selected carrier [QC].sup.+ will also be rapidly eliminated from the brain (k.sub.6 &gt;&gt;k.sub.2). Because of the facile elimination of [D-QC].sup.+ from the general circulation, only minor amounts of drug are released in the body (k.sub.3 &gt;&gt;k.sub.4); [D] will be released primarily in the brain (k.sub.4 &gt;k.sub.2). The overall result ideally will be a brain-specific sustained release of the target drug species. Specifically, Bodor et at. worked with phenylethylamine as the drug model. That compound was coupled to nicotinic acid, then quaternized to give compounds of the formula ##STR2## which were subsequently reduced by sodium dithionite to the corresponding compounds of the formula ##STR3## Testing of the N-methyl derivative in vivo supported the criteria set forth in Scheme A. Bodor et al. speculated that various types of drugs might possibly be delivered using the depicted or analogous carrier systems and indicated that use of N-methylnicotinic acid esters and amides and their pyridine ring-substituted derivatives was being studied for delivery of amino- or hydroxyl-containing drugs, including small peptides, to the brain. No other possible specific carriers were disclosed. Other reports of this work with the redox carrier system have appeared in The Friday Evening Post, Aug. 14, 1981, Health Center Communications, University of Florida, Gainesville, Fla.; Chemical & Engineering News, Dec. 21, 1981, pp. 24-25; and Science News, Jan. 2, 1982, Vol. 121, No. 1, page 7. More recently, the redox carrier system has been substantially extended in terms of possible careers and drugs to be delivered. See International Patent Application No. PCT/US83/00725, filed May 12, 1983 and published Nov. 24, 1983 under International Publication No. W083/03968. Also see Bodor et al., Pharmacology and Therapeutics, Vol. 19, No. 3, pp. 337-386 (1983); and Bodor U.S. Pat. No. 4,540,564, issued Sep. 10, 1985.
The aforementioned Bodor U.S. Pat. No. 4,540,564 specifically contemplates application of the dihydropyridine.revreaction.pyridinium salt carrier system to amino acids and peptides, particularly small peptides having 2 to 20 amino acid units. Among the amino acids and peptides mentioned in the patent are GABA, tyrosine, tryptophan, met.sup.5 -enkephalin, leu.sup.5 -enkephalin, LHRH and its analogs and others. Representative carrier-linked amino acids and peptides illustrated in the Bodor patent are the following: TBL3 - AMINO ACID/PEPTIDE CARRIER-DRUG (QUATERNARY) CARRIER-DRUG (DIHYDRO) ##STR4## ##STR5## ##STR6## NH.sub.2CH.sub.2 CH.sub.2 CH.sub.2 COOH(GABA) ##STR7## ##STR8## TyrGlyGlyPheLeu(leu.sup.5 -enkephalin) ##STR9## (not depicted)
Thus, in the depicted carrier system as applied to amino acids and peptides, the free carboxyl function is protected in an effort to prevent premature metabolism, e.g. with an ethyl ester, while the trigonelline-type carrier is linked to the amino acid or peptide through its free amino function. Oxidation of the dihydropyridine carrier moiety in vivo to the ionic pyridinium salt carrier/drug entity prevents elimination thereof from the brain, while elimination from the general circulation is accelerated, and subsequent cleavage of the quaternary carrier/drug species results in sustained delivery of the amino acid or peptide (e.g. tryptophan, GABA, leu.sup.5 -enkephalin, etc.) in the brain and facile elimination of the carrier moiety. This method is quite useful for delivery of amino acids; in the case of peptides, however, the typical suggested carboxyl protecting groups do not confer sufficient lipophilicity on the peptide molecule. Moreover, this approach does not address the problem of the enzymatic blood-brain barrier or suggest a means of avoiding that problem.
The third approach for delivering drugs to the brain using a redox system provides derivatives of centrally acting amines in which a primary, secondary or tertiary amine function has been replaced with a dihydropyridine/pyridinium salt redox system. These brain-specific analogs of centrally acting amines have been described in International Patent Application No. PCT/US85/00236, filed Feb. 15, 1985 and published Sep. 12, 1985 under International Publication No. W085/03937. The dihydropyridine analogs are characterized by the structural formula ##STR10## wherein D is the residue of a centrally acting primary, secondary or tertiary amine, and ##STR11## is a radical of the formula ##STR12## wherein the dotted line in formula (a) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (b) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system; m is zero or one; n is zero, one or two; p is zero, one or two, provided that when p is one or two, each R in formula (b) can be located on either of the two fused rings; q is zero, one, or two, provided that when q is one or two, each R in formula (c) can be located on either of the two fused-rings; and each R is independently selected from the group consisting of halo, C.sub.1 -C.sub.7 alkyl, C.sub.1 -C.sub.7 alkoxy, C.sub.2 -C.sub.8 alkoxycarbonyl, C.sub.2 -C.sub.8 alkanoyloxy, C.sub.1 -C.sub.7 haloalkyl, C.sub.1 -C.sub.7 alkylthio, C.sub.1 -C.sub.7 alkylsulfinyl, C.sub.1 -C.sub.7 alkylsulfonyl, --CH.dbd.NOR'" wherein R'" is H or C.sub.1 -C.sub.7 alkyl, and --CONR'R" wherein R' and R", which can be the same or different, are each H or C.sub.1 -C.sub.7 alkyl. These dihydropyridine analogs act as a delivery system for the corresponding biologically active quaternary compounds in vivo. Due to its lipophilic nature, the dihydropyridine analog will distribute throughout the body and has easy access to the brain through the blood-brain barrier. Oxidation in vivo will then provide the quaternary form, which will be "locked" preferentially in the brain. In contradistinction to the drug-carrier entities described in Bodor U.S. Pat. No. 4,540,564 and related publications, however, there is no readily metabolically cleavable bond between drug and quaternary portions, and the active species delivered is not the original drug from which the dihydro analog was derived, but rather is the quaternary analog itself.
The aforementioned International Publication No. WO85/03937 contemplates application of its analog system to amino acids and small peptides, e.g., the enkephalins, tryptophan, GABA, LHRH analogs and others. Illustrated redox analogs include the following: TBL3 - AMINO ACID/PEPTIDE QUATERNARY ANALOG DIHYDROPYRIDINE ANALOG ##STR13## ##STR14## ##STR15## N H.sub.2CH.sub.2 CH.sub.2 CH.sub.2 COOH(GABA) ##STR16## ##STR17## ##STR18## ##STR19## ##STR20## ##STR21## ##STR22## ##STR23##
In the depicted analog system as applied to amino acids and peptides, the free carboxyl function is thus protected to prevent premature metabolism while the dihydropyridine.revreaction.pyridinium salt type redox system replaces the free amino function in the amino acid or peptide.
As described in International Publication No. WO85/03937, the chemical processes for preparing the redox analog derivatives replace any free amino function in the selected drug with the redox analog system. When these processes are applied to amino acids, they provide a redox amino acid which no longer contains a free amino function for linkage to another amino acid or peptide via a peptide bond (--CONH--). Such an analog amino acid can thus only be used to prepare a peptide having the analog amino acid located at the peptide's N-terminus. This limits use of the redox analog amino acids in peptide synthesis. Moreover, as noted hereinabove, this approach is not designed to ultimately deliver the original peptide to the brain, since there is no cleavable bond between peptide and quaternary portions; rather, the redox portion in this approach becomes an inherent, essentially inseparable pan of a new peptide analog. Furthermore, this approach does not address the problem of the enzymatic blood-brain barrier or suggest a means for avoiding the premature degradation caused by the highly active neuropeptide degrading enzymes.
The fourth redox approach is designed to provide redox amino acids which can be used to synthesize peptides having a redox analog system inserted at a variety of locations in the peptide chain, including non-terminal positions, and has been described in Bodor U.S. Pat. No. 4,888,427, issued Dec. 19, 1989. These amino acids contain a redox system appended directly or via an alkylene bridge to the carbon atom adjacent to the carboxyl carbon. The peptides provided by U.S. Pat. No. 4,888,427 have an amino acid fragment of the formula ##STR24## incorporated therein at a non-critical position in the peptide chain, i.e., at a position which is not critical to the pharmacological effect of the peptide. In structures (A) and (B) above, Z is either a direct bond or C.sub.1 -C.sub.6 alkylene and can be attached to the heterocyclic ring via a ring carbon atom or via the ring nitrogen atom; R.sub.1 is C.sub.1 -C.sub.7 alkyl, C.sub.1 -C.sub.7 haloalkyl or C.sub.7 -C.sub.12 aralkyl when Z is attached to a ring carbon atom; R.sub.1 is a direct bond when Z is attached to the ring nitrogen atom; R.sub.2 and R.sub.3, which can be the same or different, are selected from the group consisting of hydrogen, halo, cyano, C.sub.1 -C.sub.7 alkyl, C.sub.1 -C.sub.7 alkoxy, C.sub.2 -C.sub.8 alkoxycarbonyl, C.sub.2 -C.sub.8 alkanoyloxy, C.sub.1 -C.sub.7 haloalkyl, C.sub.1 -C.sub.7 alkylthio, C.sub.1 -C.sub.7 alkylsulfinyl, C.sub.1 -C.sub.7 alkylsulfonyl, --CH.dbd.NOR'" wherein R'" is hydrogen or C.sub.1 -C.sub.7 alkyl, and --CONR'R" wherein R' and R", which can be the same or different, are each hydrogen or C.sub.1 -C.sub.7 alkyl; or one of R.sub.2 and R.sub.3 together with the adjacent ring carbon atom forms a benzene ring fused to the heterocyclic ring, which benzene ring may optionally bear one or two substituents, which can be the same or different, selected from the group consisting of hydroxy, protected hydroxy, halo, cyano. C.sub.1 -C.sub.7 alkyl, C.sub.1 -C.sub.7 alkoxy, C.sub.2 -C.sub.8 alkoxycarbonyl, C.sub.2 -C.sub.8 alkanoyloxy, C.sub.1 -C.sub.7 haloalkyl. C.sub.1 -C.sub.7 alkylthio, C.sub.1 -C.sub.7 alkylsulfinyl, C.sub.1 -C.sub.7 alkylsulfonyl, --CH.dbd.NOR'" wherein R'" is hydrogen or C.sub.1 -C.sub.7 alkyl, and --CONR'R" wherein R' and R", which can be the same or different, are each hydrogen or C.sub.1 -C.sub.7 alkyl; the dotted lines indicate that the fragment of formula (A) or (B) contains a 1,4- or 1,6-dihydropyridine, a 1,4- or 1,2-dihydroquinoline, or a 1,2-dihydroisoquinoline ring system; and wherein X.sup.- is the anion of a non-toxic pharmaceutically acceptable acid. The final redox peptide of U.S. Pat. No. 4,888,427 preferably contains a total of 2 to 20 amino acid units. Typically, except for the presence of at least one redox amino acid fragment of structure (A) or (B) and the possible protection of terminal amino and carboxyl functions, the structure of the redox peptide is identical to that of a known, naturally occurring bioactive peptide or of a known bioactive synthetic peptide (particularly one which is an analog of a naturally occurring bioactive peptide).
It is apparent from the foregoing, that the fourth redox approach, like the third approach discussed above, is not designed to ultimately deliver the original peptide to the brain because there is again no cleavable bond between peptide and quaternary portions. Again, the redox system becomes an integral part of a new peptide analog, not a means for ultimately delivering the original peptide to the brain. Still further, this approach also does not address the problem of the enzymatic blood-brain barrier or suggest a means for avoiding deactivation of the peptide by enzymes before it achieves its therapeutic objective.